RELATION OF STRUCTURE AND FUNCTION OF HU IFN-GAMMA BY SITE-DIRECTED MUTAGENESIS

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It has been shown that deletion of residue -1 to 7 or 9 residues of IFN- gamma results in the loss of biological activity. Due to the limitations of chemical modification procedures, we are using a protein engineering approach to study the relationship between the structure and function of IFN-gamma. The Hu IFN-gamma gene has been cloned into a pGitem 2 vector and pkk 223-3, pkk 233-2 expression vector. We obtained high expression of 2 forms of IFN-gamma, a truncated form and an intact form. The IFN- gamma gene was also expressed using W3110/PIIF-cye provided by Genentech, Inc. We also constructed T7 RNA polymerase/promoter system for IFN-gamma gene expression. In this system, IFN-gamma gene was expressed by infection with M 13 phage. Western Blot, SDS PAGE and antiviral activity have revealed that these cells which are induced by IPTG or IAA produced Hu IFN-gamma. We have purified the IFN-gamma by monoclonal antibody chromatography and sephacryl s-200 gel filtration. In future studies, we will make chimeric and mutant Hu IFN-gammas by PCR or site-directed mutagenesis to elucidate the relationship of structure and function of Hu IFN-gamma.